TiPS -]enuary
2994 [Vol. 251
13 be three A3 receptors: the putative A3 and
the P-site, the cloned
A3. J.
A.
RIBEIRO
AND
A.
M.
SEBASTldO
Adenosine receptor antagonists suggest the existence of adenosine A3 receptors
Lnborntq of PIf~r~flncolog!y, Gitlltctrkinrl Imtifrrtr of Scierfcc, 2781 Oeiras. Ports@.
Further evidence for adenosine A3 receptors
References
In a recent paper’, Carruthers and Fszard concisely summarized the evidence for our initial proposal for the existence of the adenosine A3 receptor*. However, in attempting to evaluate the current status of this putative A3 receptor and to compare it with the cloned A3 receptolj, several confusing statements and omissions were made. They quoted a study by Sebastiso et ~71.~ as demonstrating the presence of the putative A3 receptor in the nervous tissue. However, this paper clearly states that the inhibitory adenosine receptor in the rat hippocampus and at the neuromuscular junction is a typical A, receptor, since in both preparations the adenosine receptor antagonist DPCPX has a Ki value of approximately 0.5 nM. To provide further evidence for the present status of the A3 receptor, the authors should have mentioned that we reported that the affinities of antagonists for the A3 receptor at the frog neuromuscular junction are quite different from those for the A1 receptor5. For example, whereas at the frog neuromuscular jlmction DFCPX has a Ki value of 35 nM (Ref. 5), for the Al receptor, the Ki value is approximately 0.5~~ and for the A2 receptor it is approximately 340nM (Refs 6,7). The adenosine receptor at the frog neuromuscular junction appears to be linked to polyphosphoinositide turnover rather than inhibition of adenylate cyclase*. In addition, a recent paper characterized the putative A3 receptor in terms of radioligand binding and found that the Ki value of DPCPX for the adenosine receptor in frog brain membranes’ is similar to tha? at the frog neuromuscular junction5. This putative A3 receptor appears to be easier to identify in the frog (reviewed in Refs 10,ll) probably because of the absence of Al receptors’, but a binding site for DPCPX with a Ki value inter-
mediate to those of the A, and A2 receptor binding sites has also been identified in the rat brain”. In conclusion, the putative A3 adenosine receptor that was initially proposed by us on the basis of agonist profile*, can now be characterized by antagonist criteria. The A1 and A2 receptors were also characterized by their agonist profiles before specific antagonists became available. The most prominent feature of the putative A3 receptor is the intermediate Ki value for DPCPX; another is the very low affinity of XCC and PACPX for this receptor5e9. A final point relates to the xanthine-insensitivity of the cloned putative A3 recepto?. Traditionally, in adenosine receptor nomenclature, the common characteristic of all the adenosine receptors has been their xanthine sensitivity13. It may generate confusion if this criterion is finally laid to rest but if it is abandoned, the originally xanthine-insensitive described P-site13 should be renamed. It could possibly be called A3, since it was the third adenosine receptor to be described. There may then
1 Carruthers, A. M. and Fozard. J. R. (1993) Trrwfs Phnrt~racol. Sci. 14.290-291 2 Ribeiro, J. A. and Sebastilo, A. M. (1986) Prog. Neurobiol. 26, 179-209 3 Zhou, Q. Y. el nl. (1992) Proc. Nntl Acnd. Sci. USA 89, 7432-7436 4 SebastiBo, A. M., Stone, T. W. and Ribeiro, J. A. (ISjO) Br. /. Plrannncol. 101, 453-459 5 SebastiBo, A. M. and Ribeiro, I. A. i1989) Br. I. Plwfnncol. 96. 211-219 6
7 Lohse, M. j. et al. (1987) NRI~IYII SclznreideIwrg’s Arch. P/mmnrol. 336, 204-210 A. M. and Ribeiro, J. A. 8 Sebastilo, (1990) Br. I. Pkarmicof. 100. 55-62 9 blivrira, j. C., SebastiHo,. A. M. and Rib&o, J. A. (1993) Br. 1. Phannncol. 109, 518-523 Ribeiro, J. A. (1991) in Adeu~si~rr nud Adetkrr Nwleofides as Replafors of Cellrhr function (Phillis, J. W., ed.), pp. 155-167, CRC Press Ribeiro, J. A., Correia-de-S& I’., Cunha, R. A., bliveira. 1. C. and SebastiPo. A. M. (1991) in’ Role of Adenosine md Adenim
Nwlmtides
itI
the
Biolopicnl
Systcn~ (Imai, S. and Nakazawa,-M., eds), pp. 691-702, Elsevier Oliveira, 1. C., Sebastiio, A. M. and Ribeiro, J: A. (1991) /. Ncrtrork~m. 57, 1165-1171 Daly, J. W., Bruns, R. F. and Snyder, S. H. (1981) LifeSci.28,2083-2097
DPCPX:1,3-dipropyl-B-cyclopentylxanthine PACPX: 1,3-dipropyl-S-(2-amino-4-chlorophenyl)-xanthine XCC: 1,3-dipropyl-8-(4-carboxymethyloxyphenyl)-xanthine
Adenosine A3 receptor antagonists may be useful in the treatment of allergic discrders
Adenosine A3 receptors in mast cells We would like to add to Carruthers and Fozard’s informative article’ on the adenosine A3 receptor by noting the presence and functional correlates of this receptor in mast cells. The receptor was first described in a cultured mast cell line (RBL-2H3 cel!s) as a novel adenosme receptor with features distinct from those of the classic A, and A2 receptors. The receptor did not mediate the activation of adenylate cyclase, but instead activated phospholipase C via a G protein, inducing transient production of inositol phosphates and transient
elevation of the concentration of cytosolic Ca*+ (Ref. 2). The sensitivities of RBL-2H3 cells to various agonists and antagonists of adenosine receptors differed from those expected for the classic adenosine receptors; RBL-2H3 cells were relatively insensitive to inhibition by xanthine analogues, including various theophylline derivatives, and were activated by NECA, adenosine and R-PIA (Refs 2,3). By themselves, the adenosine analogues were weak secretagogues, but they markedly potentiated secretory responses to antigen and
probably as a Ca2 * ionophores’, consequence of a sustained activation of phospholipase D and protein kinase C. The receptor was identified as the A> receptor’ by competitive binding studies with [i’sI]APNEA, which indicated that the putative receptor had the characteristic binding profile (i.e. of the R-PIA= NECA > s-PIA) cloned adenosine Ax receptor5 and low sensitivity to xanthine antagonists. Furthermore, mRNA transcripts for A3 receptors, but not for A, or Aa receptors, were detected in poly(A’) RNA from RBL-2H3 cells. Transcripts for the Aa receptor were also detected in two other mast cell line&. The presence of A3 receptors in mast cells may provide new therapeutic opportunities for the treatment of allergic disorders. For example, treatment of RBL-2H3 cells with dexamethasone markedly increases their sensitivity to NECA such that NECA induces secretion”. If the same scenario takes place in mucosal human lung mast cells, this would have obvious c!inical relevance. This treatment would be particularly useful for the subset of asthma patients whose condition is resistant to steroid treatment if the pathogenesis of asthma involves the release of adenosine from inflammatory cells. The insensitivity of the AJ receptor to conventional xanthine antagonists provides a prospect for the design of selective inhibitors of this receptor, and the absence of Ai and A? receptors in RBL-2H3 cells makes these cells an ideal system for screening such inhibitors. The distribution of subtypes of adenosine receptors among the various species of mast cells (e.g. mucosal versus connective tissue mast cells) and basophils must also be determined to provide a rational basis for the design of therapeutic strategies and the reappraisal of previously obtained data. Differences in distribution of the receptor suhtypes might explain why adenosine analiogues suppress immunological responses of some types of mast cells and basophils (via adenylate cyciase) and potcntiate responses in other types of mast cells (via phospholipases C and D), and might also explain why mucosal mast cells are insensitive to theophylline’. Such diversity dictates that all cells should be
probed for the three receptor subtypes. MICHAEL
A.
RAMKUMAR
BEAVEN, AND
adenosine *VICKRAM ‘HYDAR
ALI
References 1 Carruthers,
265, 745-753 3 Ah, II., Muller, C. E., Daly, J. W. and Beaven, M. A. (1991) 1. Plrnriuncol. Exp. Tlrrr. 255, 954-962 4 Ramkumar, V.. Stiles, G. L., Beaven, M. A. dnd Ah, H. (1993) f. Bid. Chr. 268, 16887-16890 5 Zhou, Q-Y. et nl. (1992) Proc. Nntl Ad. Set. LISA 89, 7432-7436 6 Collado-Escobar, D., Ah, H. and Beaven, M. A. (1990) I. ftttrtzrtttof. 144, 3449-3457 7 Pearce, F. L., Befus, A. D., Gauldie, J. and Bienenstock, 1. (1982) /. Ittutttrrwl. 128, 2481-2486
APNEA: N”-2-(4-aminophenyI)ethyl-
A. M. and Fozard. J. R. (1993) Tnvtds ~~i~rt~~~c~)f. ?ic;. 14. 290-291 Ali, H.. Cunha-Melo, I. R., Saul, W. F. and Peaven, M. A. (1990) f. Biot. C/wit.
adenosine NECA: S’-N-ethyIcarboxamido-adenosine R-bk R-Nh-phenylisoproyladenosine s-PIA: s-N”-phenyIisopropy,adenosine
The cloned adenosine ASreceptor shouki take priority
armthers and
ozard reply
Part of the confusion surrounding the adenosine receptor that we designated as the ‘first’ As receptor’ arises from the fact that the number of tissues claimed to have the receptor has declined over the years. As we pointed out, the original hypothesis suggested that the first A3 receptor could mediate inhibition of central and peripheral neurotransmitter release and of the rate and force of cardiac contraction in a number of species. Ribeiro and Sebastiao conceded that the distribution of the first A3 receptor now appears to be more restricted, being definitely established only on thtl innervation of the frog sartorius muscle’ and possibly in the frog brain3. We readily concede the existence of an adenosine receptor that is neither A, nor Aa in frog tissues, but we do not agree that this vestige of the original concept should be used to perpetuate the existence of the first A3 receptor. In our view, a cloned receptor with clear functional correlates should be accepted before a putative novel receptor whose structure is unknown. Hence, we re-
main convinced that the time is right to reject the first As receptor and embrace the ‘second’ cloned A3 receptor’ whose functional importanct?“” is increasingly being recognized. Nevertheless, we do anticipate the day when the nonAl, non-AZ, non-A3 receptor that has been demonstrated so clearly by Ribeiro and Sebastiao in the frog, will be re-named and included in the classification of PI purinoceptors. A.
M.
CARRUTHERS J. R.
AND FOZARD
References 1 Carruthers, 2
3
4 5 6
A. M. and Fozard, J. R. (1993) Twttds Phtrtrtncd. Sri. 14. 290-291 Sebastiao, A. M. and Rib&o, J. A. (1989) Rr. I. Pkrtrtttttrol. 96, 211-219 Oliveira, I. C., Sehastiao. A. M. and Ribeiro, J. A. (1993) Br. I. Phnrrnwoi. 109, 518-523 Zhou, Q-Y. cf nl. (1992) Prw. Nell Acntf. Sit. !JS,i 3. 7432-7436 Fbzard, I R. and Carruthers, A. M. (I‘993) Bt. 1. P~J~irt~if?~(~f. 109, 3-5 Ramkumar, V., Stiles, G., Beaven, M. A. and Ah, H. (1993) 1. Bid. Chat. 268, 16887-16390
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